A new nanoscale drug delivery system (drug size 20-30 nm) based on naturally derived poly malic acid (PMLA) from Physarum polycephalum will be studied. Preliminary data using this system show significant anti-tumor and anti-angiogenic activity of PMLA-based drug (Polycefin) against brain cancer with no toxicity. Polycefin has a unique ability of targeting multimeric tumor-associated proteins with one drug vehicle, which is not feasible with other delivery systems. Rats with xenografted human brain tumors after a limited number of treatments with Polycefin bearing Morpholino antisense oligonucleotides to chains of vascular tumor protein, laminin-8 showed significantly increased survival (p<0.0004) and 75% decrease in tumor vascularity (p<0.001). Preliminary data show that Polycefin penetrates blood-tumor barrier (BTB) and accumulates specifically in brain and breast tumors. In this proposal, the relationship between tumor treatment efficacy and the structure of different Polycefin variants, in particular, the number and specificity of conjugated prodrugs (antisense oligonucleotides) and tumor-targeting moieties will be evaluated in vitro and in vivo. Polycefin variants will be studied in order to improve tumor delivery and the efficacy of anti-cancer drug(s). The project will focus on second-generation conjugates to treat brain gliomas and invasive breast cancer, targeting epidermal growth factor receptor (EGFR), which is overexpressed in these tumors. Additionally, our previous target, laminin-8, will be used. The ultimate goal is to select a lead vehicle-drug compound for each tumor type from these polymer-based conjugates by detailed preclinical evaluation, which would set grounds for further clinical use. To accomplish this goal, we propose: Aim 1. Structural optimization of the drug vehicle. This includes (a) improvement of endosomal disruption activity through enhanced hydrophobicity by exchange of valine in the present carrier with leucine ethylester thereby increasing membrane permeation;(b) exchanging the existing fluorescein reporter by Alexa Fluor 680 for superior imaging in vitro and in vivo. Aim 2a. Optimization of the number and relative abundance of antisense oligonucleotides on one PMLA carrier to increase the number of molecular targets to be inhibited. In vitro determination of target protein inhibition and the drug effects on tumor cell proliferation, invasion and apoptosis, to select the strongest inhibiting drug. Aim 2b. Optimization of the number and relative abundance of cell targeting monoclonal antibodies on PMLA carrier for facilitating BTB penetration and increasing tumor targeting specificity. In vitro determination of target protein inhibition and the drug effects on tumor cell proliferation, invasion and apoptosis. Aim 3. Pro-drug characterization. Determination of drug toxicity and half-life of the carrier system itself (with/without antisense oligonucleotides and monoclonal antibodies) and of the lead carrier-drug compounds. Optimization of the treatment number. Aim 4. Evaluation of the effects of lead compounds on animal survival, tumor size, vascularity, apoptosis, and expression of their molecular targets, EGFR and laminin-8, in xenotransplanted human tumors.